ABSTRACT Fractures are the most common large-organ, traumatic injuries in humans, and osteoporosis-related fractures are the fastest growing health care problem of aging. While fracture repair after surgery is usually optimal, up to 10% percent of the estimated ~8 to 10 million fractures that occur annually in the United States show delayed or impaired healing (Praemer et al., 1992). Currently, radiographic assessment, with reduction in healing complication and validated patient reported outcomes of regain of pain free weight bearing and function are the primary diagnostic tools to assess the progression of fracture healing. However none of these current assessments either define underlying biological processes that are related to the progression of fracture healing or are they prognostic for delayed healing or non-unions. Thus, there is an immense and immediate need to identify objective quantifiable biological markers: 1) that relate to the underlying biological processes of skeletal tissue healing: 2) that are indicative of the progression of skeletal tissue healing:3) that would be prognostic of deficiencies in skeletal tissue healing. The development of such an assay would be an immense benefit to: 1) be informative to the underlying causes for delayed and failed healing: 2) use in clinical trials that assess the efficacy of biological or pharmacological therapies that promote bone healing and: 3) identify those patients that would benefit from biological or pharmacological therapeutic interventions to promote bone healing. Our hypothesis is that there will be a combination of serum markers that can be used to define the biological progression of fracture healing and that we will be able to relate one or more of these markers to structural, functional and clinical characteristics that define the progression of healing. Two specific aims are proposed. Aim 1 will identify those proteins in the serum proteome that show changed levels of expression across the time course of fracture healing relative to unfractured bone. In this aim, two approaches will be used: a mass spectrometry approach and a more targeted novel aptamer-based multiplexed proteomic technology. This aim is will identify and provide preliminary quantification of a subset of proteins that can be related to various biological processes that define the temporal progression of fracture healing. In Aim 2, we will test a subset of these proteins for their statistical correlation to the development and resorption of cartilage, development and remodeling of bone and callus tissue structure mineralization as determined by both cartilage contrast enhanced and standard CT. We will test for statistical correlation of specific protein expression to specific biomechanical functions (stiffness, strength, work to failure). Finally, we will test how callus structure, function measurements and specific protein markers correlate to current clinically used Radiographic Union Score for Tibial (RUST) fracture healing (Whelan et al., 2010) that is used to assess the progression of human long bone healing. If successful this study will identify a set of proteins that can be used in a human trial to test for their diagnostic efficacy to follow fracture healing and their prognostic efficacy for delayed or failed healing.